The probe was stepped up earlier today amid growing concerns for the 24-year-old's welfare, as sniffer dogs, forensic teams and specialist divers scoured areas in the north-east of the city.
Ms Buckley was last seen more than three days ago leaving a nightclub with Alexander Pacteau in the early hours of Sunday morning. She reportedly left the property at 4am to walk home and has not been seen since.
This evening, police said they were now following a 'definite line of enquiry'.
Forensics teams searched around the flat where she was last seen today as concerns grew about her safety
With the time since Miss Buckley went missing passing 70 hours, police stepped up the search this afternoon
Lines of police officers today combed a park in the north-east of the city where her handbag was later found by a member of the public and divers searched the River Kelvin which borders the area.
Forensics officers meanwhile examined the rooms and garden of the flat of Mr Pacteau, with whom she left the nightclub shortly after midnight on Saturday.
Police earlier ruled him out as a suspect but a large-scale search was carried out around his home as officers searched for clues.
A police helicopter meanwhile circled between the villages of Milngavie and Drymen to the north of Glasgow, where a grey car linked to the search was seen on Monday.
A Police Scotland spokesman said today: 'The search resumed at first light this morning with specialist search officers and air support in the area of Dawsholm Park and the north-west of Glasgow.'
Police activity has centred on two locations; Dawsholm Park, where the black handbag she was carrying on the night of her disappearance was found, and around a flat in Dorchester Avenue where she went with Mr Pacteau.
Mr Pacteau, who police said earlier is not being treated as a suspect, told officers the pair travelled to his flat before she left the property at around 4am to return to her home four miles across the city.
Relatives of Mr Pacteau yesterday confirmed 24-year-old Miss Buckley went to his flat from a nightclub before she disappeared.
Mr Pacteau's mother Noreen, 43, told the Daily Record: 'She left his property at four o'clock in the morning and she was seen by other witnesses, not Alexander. So he is not a suspect.'
Miss Buckley left a Glasgow's The Sanctuary nightclub in the early hours of Sunday morning without collecting her coat after telling friends she was going to the toilet.
Friends say she had been drinking but was not drunk.
Karen Buckley, 24, pictured (far left) with friends on the night she went missing
CCTV cameras filmed her talking to a man, believed to be Mr Pacteau, outside the club before getting into a car and travelling to his flat in nearby Kelvindale.
Police said the pair were 'intimate' at his flat. She later left but did not turn up at her own home which is three miles away across the city.
Miss Buckley, who is brunette and 5ft 2in, was wearing a black jumpsuit and red heels when she was last seen leaving Mr Pacteau's flat in Kelvindale at around 4am.
Her handbag was found in a park not far from Mr Pacteau's home yesterday, causing police to say they are 'gravely concerned' about her safety.
The family of Miss Buckley, who is from Cork in the Republic of Ireland, have flown to Scotland to help with the search for the Glasgow Caledonian University student, who studies occupational therapy.
Her mother Marion told a press conference in Glasgow: 'We just want Karen home safely, we are desperate. She is our only daughter, we love her dearly.
'If anybody has any information please come forward, we would dearly appreciate it.'
Her father John Buckley, 62, added: 'We are extremely concerned for her. We are desperate to get her back and safe with her family - she is our only daughter, we love her dearly and just want her to come home safe and sound.
'She always keeps in contact with her family and friends to let them know where she is and what she is up to.
'To not return to her flat or be in contact with her pals - not responding to texts and calls to her mobile is very, very worrying as it is so out of character.'
Detective Superintendent Jim Kerr, from Police Scotland's major investigations teams, stressed at a press conference yesterday that the man since identified as Mr Pacteau is 'not a suspect'.
Mr Kerr said: 'We've traced the man she was with in Dorchester Avenue. He believes he was intimate with her at his flat consensually in the early hours of Sunday.'
The police officer added: 'From what we can see, she does not appear to be under duress, there's no signs of a struggle or reluctance on her part to leave the club.
Police are also keen to find out more about a grey car that was seen on the roads between Milngavie and Drymen north of Glasgow between 11am and 3pm on Monday.
Mr Kerr said: 'The activity of this grey car on the Monday is something that we're a wee bit concerned about. This car has been seen at various locations on these roads and I want to know why.'
Speaking of Miss Buckley, Mr Kerr added: 'We know she left the club with her handbag – which has been found - but left her coat behind and so far has not returned to pick it up.
'Why she left so suddenly without telling her friends we just don't know – it was very much out of character – she is usually very good at keeping in touch with pals.'
He added: 'We are gravely concerned that Karen has come to some harm, whether that is down to foul play, criminality or she has taken unwell or had an accident is obviously still to be established.'
Meanwhile, in Ms Buckley's home in north Cork a special prayer service was arranged for local people to show support for the family.
The private Mass of Hope, as it was being described, was being held tonight at the Church of Saint Michael The Archangel, Mourneabbey, near the town of Mallow.
Father Joe O'Keeffe, the parish priest, appealed for parishioners and friends of the Buckley family to be given the space to offer prayers for the family this evening.
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